Abstract
Foot and mouth disease induced by foot and mouth disease virus (FMDV) is severe threat to cloven-hoofed domestic animals. The gene 3Dpol in FMDV genome encodes the viral RNA polymerase, a vital element for FMDV replication. In this study, a conserved 3D-7414shRNA targeting FMDV-3Dpol gene was designed and injected into pronuclear embryos to produce the transgenic goats. Sixty-one goats were produced, of which, seven goats positively integrated 3D-7414shRNA. Loss of function assay demonstrated that siRNA effectively knockdown 3Dpol gene in skin epithelium cells of transgenic goats. Subsequently, the tongue epithelium cells from transgenic and non-transgenic goats were infected with FMDV O/YS/CHA/05 strain. A significant decrease of virus titres and virus copy number was observed in cells of transgenic goats compared with that of non-transgenic goats, which indicated that 3D-7414siRNA inhibited FMDV replication by interfering FMDV-3Dpol gene. Furthermore, we found that expression of TLR7, RIG-I and TRAF6 was lower in FMDV infected cells from transgenic goats compared to that from non-transgenic goats, which might result from lower virus copy number in transgenic goats’ cells. In conclusion, we successfully produced transgenic goats highly expressing 3D-7414siRNA targeting 3Dpol gene, and the tongue epithelium cells from the transgenic goats showed effective resistance to FMDV.
Highlights
Goat, as an economic farm animal in the world, can provide ~6.6 million meat product and 1.18 million tonnes fresh furs for human consumption (FAO, 2010)
We investigated the expression of two pattern recognition receptors (PRRs), Toll-like receptor 7(TLR7) and Retinoic acid-inducible gene-I (RIG-I) as well as their downstream gene (Tumour necrosis factor receptor-associated factor 6, TRAF6) in epithelium cells between transgenic goats (Tg) and non-transgenic goats (NTg) after treated with 100TCID50 foot and mouth disease virus (FMDV)
Our results showed that expression of TLR7 was lower in epithelium cells from transgenic goats than that from non-transgenic goats at 12 and 24hpi
Summary
As an economic farm animal in the world, can provide ~6.6 million meat product and 1.18 million tonnes fresh furs for human consumption (FAO, 2010). Large amount of studies indicate that RNA interference (RNAi) is an effective approach in interfering virus replication[10]. Virus replication is usually disrupted by transfecting shRNA or shRNA expressing plasmid in RNAi-mediated cell or mouse models[10,11,12,13]. Transfection of shRNA targeting 3D and 2B can effectively inhibit FMDV replication, and the transferred exogenous gene can stably be delivered to offspring[16,17]. We produced transgenic goats with high expression of siRNA targeting 3Dpol gene, and investigated differential disease resistance between transgenic goats (Tg) and non-transgenic goats (NTg) by infecting tongue epithelial cells with FMDV, which provided a model to explore the role of transgenic animals in disease resistance
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